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Biochemistry 2009, 48, 8006–8013 DOI: 10.1021/bi901064k
An Examination of the Relationship between Active Site Loop Size and Thermodynamic Activation Parameters for Orotidine 50 -Monophosphate Decarboxylase from Mesophilic and Thermophilic Organisms† Krisztina Toth,‡ Tina L. Amyes,‡ B. McKay Wood,§ Kui K. Chan,§ John A. Gerlt,§ and John P. Richard*,‡ ‡
Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260-3000, and §Departments of Biochemistry and Chemistry, University of Illinois, Urbana, Illinois 61801 Received June 23, 2009; Revised Manuscript Received July 19, 2009
Closure of the active site phosphate gripper loop of orotidine 50 -monophosphate decarboxylase from Saccharomyces cerevisiae (ScOMPDC) over the bound substrate orotidine 50 -monophosphate (OMP) activates the bound substrate for decarboxylation by at least 104-fold [Amyes, T. L., Richard, J. P., and Tait, J. J. (2005) J. Am. Chem. Soc. 127, 15708-15709]. The 19-residue phosphate gripper loop of the mesophilic ScOMPDC is much larger than the nine-residue loop at the ortholog from the thermophile Methanothermobacter thermautotrophicus (MtOMPDC). This difference in loop size results in a small decrease in the total intrinsic phosphate binding energy of the phosphodianion group of OMP from 11.9 to 11.6 kcal/mol, along with a modest decrease in the extent of activation by phosphite dianion of decarboxylation of the truncated substrate 1-(β-D-erythrofuranosyl)orotic acid. The activation parameters ΔHq and ΔSq for kcat for decarboxylation of OMP are 3.6 kcal/mol and 10 cal K-1 mol-1 more positive, respectively, for MtOMPDC than for ScOMPDC. We suggest that these differences are related to the difference in the size of the active site loops at the mesophilic ScOMPDC and the thermophilic MtOMPDC. The greater enthalpic transition state stabilization available from the more extensive loop-substrate interactions for the ScOMPDC-catalyzed reaction is largely balanced by a larger entropic requirement for immobilization of the larger loop at this enzyme. ABSTRACT:
Orotidine 50 -monophosphate decarboxylase (OMPDC)1 is a remarkable enzyme because it employs no metal ions or other cofactors but yet effects an enormous ca. 30 kcal/mol stabilization of the transition state for the chemically very difficult decarboxylation of orotidine 50 -monophosphate (OMP) to give uridine 50 -monophosphate (UMP) (Scheme 1) (1-3). The enormous 1017-fold rate acceleration for decarboxylation of enzyme-bound OMP [kcat = 15 s-1 (4)] is a consequence of the exceedingly slow decarboxylation of OMP in water [t1/2 ≈ 78 million years (3)] through an unstable vinyl carbanion intermediate. This remarkable efficiency led to the proposal of several different mechanisms for the OMPDC-catalyzed reaction that avoid formation of an unstable carbanion (5-10). However, we recently showed that OMPDC meets its catalytic challenge head-on by stabilizing an enzyme-bound vinyl carbanion (Scheme 1) (11, 12). † This work was supported by Grants GM39754 to J.P.R. and GM65155 to J.A.G. from the National Institutes of Health. *To whom correspondence should be addressed. Telephone: (716) 645-4232. Fax: (716) 645-6963. E-mail:
[email protected]. 1 Abbreviations: OMP, orotidine 50 -monophosphate; UMP, uridine 50 -monophosphate; OMPDC, orotidine 50 -monophosphate decarboxylase; ScOMPDC, orotidine 50 -monophosphate decarboxylase from Saccharomyces cerevisiae (yeast); EcOMPDC, orotidine 50 -monophosphate decarboxylase from Escherichia coli; MtOMPDC, orotidine 50 -monophosphate decarboxylase from Methanothermobacter thermautotrophicus; EO, 1-(β-D-erythrofuranosyl)orotic acid; EU, 1-(β-D-erythrofuranosyl)uridine; DHAP, dihydroxyacetone phosphate; TIM, triosephosphate isomerase; GPDH, glycerol-3-phosphate dehydrogenase; MOPS, 3-(N-morpholino)propanesulfonic acid; IPTG, isopropyl β-D-thiogalactopyranoside; NADH, nicotinamide adenine dinucleotide, reduced form; PDB, Protein Data Bank.
pubs.acs.org/Biochemistry
Published on Web 07/20/2009
One of the principal differences between catalysis by enzymes and by small molecules is that enzymes have evolved unique mechanisms to utilize binding interactions with nonreacting portions of the substrate for transition state stabilization (13). We have shown that the binding of phosphite dianion to OMPDC from Saccharomyces cerevisiae (yeast, ScOMPDC) results in an 80000-fold increase in the second-order rate constant (kcat/Km) for enzyme-catalyzed decarboxylation of the truncated substrate 1-(β-D-erythrofuranosyl)orotic acid (EO), which lacks a 50 -phosphodianion moiety, to give 1-(β-D-erythrofuranosyl)uridine (EU) (Scheme 2) (14). This shows that the nonreacting phosphodianion group of OMP does not function simply to anchor the substrate to the enzyme but rather serves the more important role of activating the enzyme toward decarboxylation of bound OMP. Similar experiments provided evidence that the “intrinsic phosphate binding energy” of the substrate phosphodianion group of dihydroxyacetone phosphate (DHAP) is utilized in stabilization of the transition state of the aldose-ketose isomerization reaction catalyzed by triosephosphate isomerase (TIM) (15, 16) and of the hydride transfer reaction catalyzed by glycerol-3-phosphate dehydrogenase (GPDH) (Scheme 3) (17). OMPDC (10, 18-22), TIM (23-25), and GPDH (26, 27) each have a flexible “phosphate gripper” loop that is open at the free enzyme but closes over the phosphodianion group of the bound substrate to sequester the substrate from bulk solvent. We have proposed that this phosphate-driven loop closure reduces the barrier for reaction at the loop-closed compared with the loop-open enzymes, and that it is the underlying origin of the r 2009 American Chemical Society
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Scheme 1
Scheme 2
enzyme-bound OMP such that MtOMPDC pays a substantially smaller entropic price, but a larger enthalpic price, for conversion of the Michaelis complex to the transition state for decarboxylation than ScOMPDC does. EXPERIMENTAL PROCEDURES
Scheme 3
104-106-fold effect of the nonreacting substrate phosphodianion group on the chemical reactivity of the enzyme-bound substrate (17). The phosphate gripper loops of ScOMPDC and of OMPDC from Escherichia coli (EcOMPDC) both extend 19 residues, from the strictly conserved Pro-202 to Val-220 for ScOMPDC (21) (Figure 1) and from Pro-189 to Pro-207 for EcOMPDC (19, 20). By contrast, the corresponding loop of OMPDC from the thermophile Methanothermobacter thermautotrophicus (MtOMPDC) extends only nine residues, from Pro-180 to Asp-188 (22) (Figure 1). These three enzymes show an otherwise high degree of structural homology (18) which then raises the question of whether there may be an underlying mechanistic imperative for the difference in the size of the flexible loops for enzymes from mesophiles and thermophiles. For example, if the size of this loop is related to catalytic efficiency, then the smaller loop for MtOMPDC might be expected to play a reduced role in promoting decarboxylation through utilization of the binding energy of the nonreacting substrate phosphodianion group of OMP or of a phosphite dianion activator. We report here the results of experiments that were designed to compare the intrinsic phosphate binding energy and the thermodynamic activation parameters for OMPDCs from mesophilic and thermophilic organisms. The thermophilic enzyme with the shorter phosphate gripper loop, MtOMPDC, shows an intrinsic phosphate binding energy of the phosphodianion group of OMP and phosphite activation of decarboxylation of the truncated substrate EO that are similar to those observed for the mesophilic ScOMPDC. By contrast, there is a significant difference in the activation parameters ΔHq and ΔSq for the decarboxylation of
Materials. Orotidine 50 -monophosphate trisodium salt (99%) was purchased from Sigma or was prepared by chemical or enzymatic methods from uridine 50 -monophosphate using modifications of literature procedures (28-30). 1-(β-D-Erythrofuranosyl)orotic acid (EO) and 1-(β-D-erythrofuranosyl)uridine (EU) were available from an earlier study (14). Sodium phosphite (dibasic, pentahydrate), 3-(N-morpholino)propanesulfonic acid (MOPS, g99.5%), and ammonium acetate (g99%) were purchased from Fluka. Water was from a Milli-Q Academic purification system. All other chemicals were reagent grade or better and were used without further purification. Preparation of OMPDCs. The C155S mutant OMPDC from S. cerevisiae (ScOMPDC) was prepared as described previously (31, 32). This mutant is more stable than, but kinetically and structurally essentially identical with, wild-type yeast OMPDC (33). Wild-type OMPDC from M. thermautotrophicus (MtOMPDC) was prepared as described previously (32). The gene for wild-type OMPDC from E. coli (EcOMPDC) was cloned from E. coli K12 genomic DNA by W. Shan Yew, and the protein was expressed in E. coli BL21(DE3) using a modified pET-15b plasmid with a His10 tag, as described previously for OMPDCs with a His6 tag (32). The cells were grown at 37 °C, induced with IPTG (0.5 mM) when the cell density reached an OD600 of 0.6, and harvested after 18 h. Purification was conducted as described previously for ScOMPDC and MtOMPDC (32) except that no Q-Sepharose column was used. The protein was dialyzed at 5 °C against storage buffer [20 mM Tris-HCl (pH 7.0), 100 mM NaCl, and 20% glycerol], concentrated to ca. 25 mg/mL via ultrafiltration, flash-frozen in liquid nitrogen as 25 μL pellets, and stored at -80 °C. Preparation of Solutions. Solution pH was determined at 25 °C using an Orion model 720A pH meter equipped with a Radiometer pHC4006-9 combination electrode that was standardized at pH 7.00 and 10.00 at 25 °C. Stock solutions of OMP were prepared in water, and the OMP concentration was determined from the absorbance in 0.1 M HCl at 267 nm using an ε of 9430 M-1 cm-1 (34). Stock solutions of 1-(β-D-erythrofuranosyl)orotic acid (EO) were prepared and neutralized to pH ≈6 as described previously (14, 31), and the EO concentration was determined from the absorbance in 0.1 M HCl at 267 nm using an ε of 9570 M-1 cm-1 reported for orotidine (34). The stock solution of phosphite (100 mM, 80% free base, I = 0.28) was prepared by addition of a measured amount of 1 M HCl to the sodium salt to give the desired acid/base ratio. MOPS buffers were prepared by addition of measured amounts of 1 M
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FIGURE 1: Comparison of the closed active site loops of the dimeric OMPDCs from S. cerevisiae [19 residues, left structure, PDB entry
3GDL (46)] and M. thermautotrophicus [nine residues, right structure, PDB entry 3G1A (46)] liganded with 6-azauridine 50 -monophosphate. The active site loops are colored red, and the remainder of the monomer is colored gray. The second monomer is colored cyan (ScOMPDC) or magenta (MtOMPDC). The loop for ScOMPDC extends from Pro-202 to Val-220, and the loop for MtOMPDC extends from Pro-180 to Asp-188. The images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by National Institutes of Health Grant P41 RR-01081).
HCl and solid NaCl to give the desired acid/base ratio and ionic strength. Samples of OMPDCs that had been stored at -80 °C were defrosted and dialyzed at 4 °C against 10 mM MOPS (50% free base) (pH 7.1) containing 100 mM NaCl, unless noted otherwise. Determination of Values of kcat and kcat/Km for Turnover of OMP by OMPDCs at Various Temperatures. All assays were conducted in 10 mM MOPS (50% free base) at pH 7.1 and I = 0.105 (NaCl) using a Cary 3E spectrophotometer equipped with a temperature-controlled Peltier block multicell changer. The temperature dependence of the difference between the extinction coefficients of OMP and UMP at 279 nm, Δε (M-1 cm-1), was determined using the following procedure. First, the spectrophotometer was zeroed at 279 nm using a solution of 10 mM MOPS at pH 7.1 and I = 0.105 (NaCl) at 25 °C. A small aliquot of OMP was added to give a final concentration of 100 μM OMP, and the absorbance of the resulting solution was determined at several temperatures between 10 and 75 °C. The temperature was then returned to 25 °C, and 1 μL of a solution of ScOMPDC was added to give a final enzyme concentration of 21 nM. The ensuing decarboxylation of OMP was allowed to proceed to completion to give a quantitative yield of UMP, and the final absorbance of the solution was again determined at several temperatures between 10 and 75 °C. The observed absorbance changes were used to calculate the following values of Δε (M-1 cm-1): 10 °C, 2290; 25 °C, 2400 (4); 35 °C, 2490; 45 °C, 2560; 55 °C, 2610; 65 °C, 2660; and 75 °C, 2720. The following values of Δε at other temperatures were obtained by extrapolation or interpolation using the two linear regions of the biphasic linear correlation between Δε (M-1 cm-1) and T: 5 °C, 2250; 12 °C, 2300; 15 °C, 2330; and 17 °C, 2340. For the determination of values of kcat, the reaction mixtures (1 mL total) containing buffer and OMP ([OMP]o = 50-250 μM, .10Km) were equilibrated at the temperature of interest and the reaction was initiated by the addition of 1-3 μL of a stock solution of OMPDC using a cuvette admixer without removal of the cuvette from the temperature-controlled block. The initial velocity of decarboxylation of OMP under these conditions, Vmax (M s-1), was determined within 1 min by monitoring the decrease in absorbance at 279 nm using the appropriate value of Δε (M-1 cm-1) for the temperature of interest. The observed values of Vmax were shown to be proportional to enzyme concentration in the following ranges: ScOMPDC, 11-42 nM; MtOMPDC, 76-150 nM; and EcOMPDC, 30-230 nM. At some temperatures, the use of lower concentrations of OMPDC
resulted in reduced activity, presumably as a result of dissociation of the active dimer to give the inactive monomeric form (4). These conditions were 38 nM MtOMPDC at 45 or 55 °C, where kcat was reduced by 20%; 30 nM EcOMPDC at 10 °C, where kcat was reduced by 30% (the data were therefore obtained using 60 nM enzyme); 30 nM EcOMPDC at 5 °C, where kcat was reduced by 50% (the data were therefore obtained using 90-230 nM enzyme); and 15 nM EcOMPDC at 25 °C, where kcat was reduced by 20%. To verify that the temperature variation did not result in a loss of enzyme activity in the time frame of the assays which were conducted over a period of e1 min (see above), periodic controls were conducted in which the stock solution of OMPDC was incubated in a water bath at the assay temperature of interest for 1-5 min prior to its use in the assay at this temperature. It was found that this preincubation did not significantly affect the observed activity at the temperature of interest. Values of kcat (s-1) were calculated from the values of Vmax (M s-1) using eq 1. In all cases, the concentration of ScOMPDC in the stock solution was calculated from the values of Vmax (M s-1) determined in side-by-side standard assays at 25 °C using eq 1 with a kcat of 15 s-1 (4). For determination of the values of kcat at 25 °C for MtOMPDC (4.7 s-1) and EcOMPDC (13 s-1), the concentration of OMPDC was determined from the absorbance of the protein at 280 nm in 10 mM MOPS (50% free base) at pH 7.1 containing 100 mM NaCl and extinction coefficients of 6100 M-1 cm-1 (MtOMPDC) and 10100 M-1 cm-1 (EcOMPDC) that were calculated using the ProtParam tool available on the ExPASy server (35, 36). For determination of the values of kcat at other temperatures, the concentrations of MtOMPDC and EcOMPDC were determined from the values of Vmax (M s-1) determined in side-by-side standard assays at 25 °C using eq 1 with kcat values of 4.7 and 13 s-1, respectively. kcat ¼ Vmax =½E
ð1Þ
Values of kcat/Km for turnover of OMP by ScOMPDC and MtOMPDC at various temperatures were determined in experiments in which the complete disappearance of a relatively low initial concentration OMP was monitored at 279 nm. Reactions (1 mL total, [OMP]o = 4-40 μM) were initiated by the addition of OMPDC as described above to give a final concentration of 20-70 nM ScOMPDC or 50-420 nM MtOMPDC. The observed rate constants kobsd (s-1) for the first-order decay of OMP in the final stages of the reactions where [OMP]t e 0.30.5Km were obtained from the fits of the absorbance versus time data to a single exponential. Experiments were conducted at
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Table 1: Kinetic Parameters and Intrinsic Phosphate Binding Energies for Decarboxylation of OMP and EO Catalyzed by ScOMPDC and MtOMPDC and for the Phosphite-Activated Reactions of EO at pH 7.0 and 25 °C kcat/Km (M-1 s-1)
ScOMPDC MtOMPDC
OMPa
EO
IPBEb (kcal/mol)
(kcat/Km)E 3 HPi/Kdc (M-2 s-1)
phosphite activationd (M-1)
1.1 107 3.1 106
2.1 10-2e 8.7 10-3
11.9 11.6
11700e 2500
5.6 105 2.9 105
a Data at pH 7.1 from Table 2. b Intrinsic phosphate binding energy calculated from the ratio of the second-order rate constants for the reactions of OMP and EO. c Third-order rate constant for phosphite-activated decarboxylation of EO catalyzed by OMPDC, calculated as the slope of the plot of (kcat/Km)obsd vs [HPO32-] at low concentrations of phosphite (Figure 2). d Ratio of the third-order rate constant for the phosphite-activated decarboxylation of EO and the second-order rate constant for the unactivated decarboxylation of EO. e Data at pH 7.1 from ref 14.
several enzyme concentrations in the indicated ranges to ensure that the observed first-order decay represents enzyme-catalyzed reaction of OMP rather than a loss of enzyme activity with time, and the values of kobsd were shown to be proportional to [E]. Values of kcat/Km (M-1 s-1) were then calculated using the relationship kcat/Km = kobsd/[E], and values of Km for OMP were obtained by combining the experimental values of kcat (s-1) and kcat/Km (M-1 s-1) (see Table 1). In all cases, the concentration of OMPDC was determined from the values of Vmax (M s-1) determined in side-by-side standard assays at 25 °C using eq 1 with the appropriate value of kcat. This analysis is possible because the initial concentration of OMP in these experiments ([OMP]o e 40 μM) was chosen (see below) such that the amount of the UMP product generated (e40 μM) is insufficient to result in significant inhibition of the OMPDC of interest. The initial concentration of OMP ([OMP]o) was chosen on the basis of the following: For ScOMPDC, a Ki value of 400 μM has been reported for binding of UMP to ScOMPDC at 25 °C under our experimental conditions (4), and there was no significant change in the value of Km determined in our experiments at 45 °C for an [OMP]o of 11-33 μM. For MtOMPDC at 25 °C, experiments conducted at an [OMP]o of 200-400 μM yielded apparent values of Km that are consistent with values of Ki for binding of UMP to MtOMPDC of ca. 100 μM at 25 °C and ca. 200 μM at 55 °C. There was no significant change in the value of Km determined in our experiments when the initial concentration of OMP was varied in the range of 6-30 μM at 5 °C, 4-12 μM at 25 °C, 1222 μM at 35 °C, or 18-39 μM at 55 °C. Turnover of EO by MtOMPDC in the Absence and Presence of Phosphite Dianion. The decarboxylation of the truncated substrate 1-(β-D-erythrofuranosyl)orotic acid (EO, 5 mM) in 50 mM MOPS (45% free base) at pH 7.0, 25 °C, and I = 0.14 (NaCl) catalyzed by MtOMPDC (350 μM) was followed in a discontinuous assay in which the initial velocity of formation of the product 1-(β-D-erythrofuranosyl)uridine (EU) was monitored by HPLC. For these experiments, MtOMPDC was dialyzed at 4 °C against 100 mM MOPS (45% free base) at pH 7.0 and I = 0.28 (NaCl). The reaction in a total volume of 200 μL was initiated by the addition of EO to a solution of the enzyme in buffer that was equilibrated at 25 °C. The reaction was followed for ca. 7 h, during which time there was 8% reaction of EO. At various times, an aliquot (20 μL) was withdrawn and quenched to pH 3.8 by the addition of 180 μL of ice-cold 5 mM formic acid. The enzyme was removed by ultrafiltration using an Amicon Microcon filtration device (10K molecular weight cutoff), and the filtrate (150 μL) was analyzed by HPLC using a using a Waters Atlantis dC18 3 μm column (3.9 mm 150 mm) with an isocratic flow of 10 mM NH4OAc (pH 4.2) at a rate of 1 mL/min and peak detection at 262 nm. Under these conditions, the unreacted EO eluted close to the void volume and the product
EU eluted at ca. 7 min. The concentration of the product EU in the reaction mixture at time t, [EU]t, was obtained from the HPLC peak area by interpolation of a standard curve that was constructed using authentic EU. The concentration of MtOMPDC in the reaction mixture was determined by a periodic standard assay (see above), and it was shown that there was no significant decrease in enzyme activity during the reaction. The initial velocity of the reaction, vi (M s-1), was determined as the slope of the linear plot of [EU]t versus time. The second-order rate constant, (kcat/Km)o (M-1 s-1), for MtOMPDC-catalyzed decarboxylation of EO was calculated using the relationship (kcat/Km)o = vi/[E][S]o. The decarboxylation of EO (0.12 mM) in the presence of 236 mM phosphite dianion and 5 mM MOPS at pH 7.0, 25 °C, and I = 0.14 (NaCl) catalyzed by MtOMPDC (22-38 μM) was monitored spectrophotometrically at 283 nm. Reactions (1 mL total volume) were initiated by the addition of 50 μL of a solution of MtOMPDC in 100 mM MOPS [pH 7.0 and I = 0.28 (NaCl)] and were monitored for up to 6 h. The concentration of MtOMPDC in the reaction mixture was determined by a standard assay, and it was shown that there was no significant decrease in enzyme activity during the reaction. These reactions obeyed excellent first-order kinetics with stable end points, and values of kobsd (s-1) for the reaction of EO were obtained from the fits of the absorbance versus time data to a single exponential. The apparent second-order rate constants, (kcat/Km)obsd (M-1 s-1), for MtOMPDC-catalyzed decarboxylation of EO at the various concentrations of phosphite dianion were calculated using the relationship (kcat/Km)obsd = kobsd/[E]. RESULTS The second-order rate constant for decarboxylation of the truncated substrate 1-(β-D-erythrofuranosyl)orotic acid (EO) lacking a 50 -phosphodianion group catalyzed by OMPDC from M. thermautotrophicus (MtOMPDC) at pH 7.0, 25 °C, and I = 0.14 (NaCl) was determined as (kcat/Km)o = 8.7 10-3 M-1 s-1 (Table 1). Figure 2 shows the dependence of the apparent second-order rate constant (kcat/Km)obsd for decarboxylation of the truncated substrate EO catalyzed by MtOMPDC on the concentration of added phosphite dianion at pH 7.0, 25 °C, and I = 0.14 (NaCl). There is no evidence for saturation of MtOMPDC by phosphite dianion, and the data for
